157

307

Chapter 27: Amino Acids, Peptides, and Proteins.monomer unit: α-amino acids

Biopolymer: the monomeric amino acids are linked through an amide bond (the carboxylic acids of one AA with the α-amino group of a second)

peptide (< 50 amino acids)protein (> 50 amino acids)

R CO2H

NH2H

!- Amino Acid

R = sidechain

R1

H3N CO2

R2

H3N CO2

+ - H2OR1

H3N

HN

O

CO2

R2

C-terminus

N-terminus

Peptide or protein (polypeptide)

NH

O

R3

HN

O

R2

NH

O

R1

HN

NH

O

R7

HN

O

R6

NH

O

R5

R4 HN

O

308

27.1: Classification of Amino Acids. AA’s are classified according to the location of the amino group.

There are 20 genetically encoded α-amino acids found in peptides and proteins

19 are primary amines, 1 (proline) is a secondary amine19 are “chiral”, 1 (glycine) is achiral; the natural configuration of the α-carbon is L.

!-amino acid(2-amino carboxylic acid)

C CO2HH2N

H

H

C CO2HC

H

H

H2N

H

H

C CC

H

H

H2N

H

H

CO2H

H

H

"-amino acid(3-amino carboxylic acid)

#-amino acid(4-amino carboxylic acid)

CO2H

CH3

H2N H

CO2H

R

H2N H

L-alanine

CHO

HO H

H OH

CH2OH

CHO

H OH

HO H

CH2OH

L-erythroseD-erythrose

CO2H

H2N H

H OH

CH3

CO2H

H2N H

H3C H

CH2CH3

L-theronine(2S,3R)

L-isoleucine(2S,3S)

CHO

CH2OH

H OH

D-glyceraldehyde

CHO

CH2OH

HO H

L-glyceraldehyde

158

309

α-Amino acids are classified by the properties of their sidechains. Nonpolar:

(S)-(+)-Valine (Val, V)

(S)-(–)-Tryptophan (Trp, W)(S)-(–)-Proline (Pro, P) (S)-(–)-Phenylalanine (Phe, F)

COO–

NH3

H

N

H

COO–

NH3

COO–

NH3

COO–

NH3

NH3

COO–

COO–

(2S,3S)-(+)-Isoleucine (Ile, I)

(S)-(+)-Alanine (Ala, A)

(S)-(–)-Leucine (Leu, L)

Glycine (Gly, G)

NH3

COO–

COO–

NH3NH

NH3

COO–S

(S)-(–)-Methionine (Met, M)

Polar but non-ionizable:

(S)-(+)-Glutamine (Gln, Q)

H2N

O

NH3

COO–

(S)-(–)-Tyrosine (Tyr, Y)(2S,3R)-(–)-Threonine (Thr, T)(S)-(–)-Serine (Ser, S)

COO–

NH3

COO–

NH3

COO–

NH3 NH3

COO–

NH3

COO–

O

H2NHS

HO

OH

HO

(R)-(–)-Cysteine (Cys, C) (S)-(–)-Asparagine (Asn, N)

pKa ~ 13 pKa ~ 13

pKa ~ 8.2

pKa ~ 10.1

310

Acidic:

Basic:

(S)-(–)-Histidine (His, H)

NH3

COO–COO–

NH3 NH3

COO–N

H

NN

H

H

(S)-(+)-Lysine (Lys, K) (S)-(+)-Arginine (Arg, R)

pKa ~ 10.5 pKa ~ 6.0 pKa ~ 12.5

H2N

NH H

H3N

-O

O NH3

COO–

(S)-(+)-Aspartic Acid (Asp, D)

COO–

NH3

O

-O

(S)-(+)-Glutamic Acid (Glu, E)

pKa ~ 3.6 pKa ~ 4.2

27.2: Stereochemistry of Amino Acids: The natural configuration of the α-carbon is L. D-Amino acids are found in the cell walls of bacteria. The D-amino acids are not geneticallyencoded, but derived from the epimerization of L-isomers

159

311

27.3: Acid-Base Behavior of Amino Acids. Amino acids exist as a zwitterion: a dipolar ion having both a formal positive and formal negative charge (overall charge neutral).

CO2H

R

H

H2N CO2

R

H

H3N_+

Amino acids are amphoteric: they can react as either an acid or abase. Ammonium ion acts as an acid, the carboxylate as a base.

Isoelectric point (pI): The pH at which the amino acid exists largely in a neutral, zwitterionic form (influenced by the nature of the sidechain)

pKa ~ 5 pKa ~ 9

CO2

R

H

H3N_+CO2H

R

H

H3N+

CO2

R

H

H2NHO

_

pKa2low pH high pH

_H3O+

pKa1

Table 27.2 (p. 1115) & 27.2 (p. 1116)

312

pI =pKax + pKay

2

CO2

CH3

H

H3NCO2H

CH3

H

H3N

low pH

CO2

CH3

H

H2N+ +

high pH

pKa1(2.3)

pKa2(9.7)

CO2H

CH2

H

H3N

CO2H

pKa3(3.6)

CO2

H

H3N

CH2

CO2H

CO2

H

H3N

CH2

CO2

pKa2(9.6)

pKa1(1.9)

CO2

H

H2N

CH2

CO2

low pH high pH

CO2H

(CH2)4

H

H3N

NH3

pKa2(9.0)

CO2

H

H3N

(CH2)4

NH3

CO2

H

H2N

(CH2)4

NH3

pKa3(10.5)

pKa1(2.2)

CO2

H

H2N

(CH2)4

NH2

low pH high pH

160

313

Electrophoresis: separation of polar compounds based on theirmobility through a solid support. The separation is based on charge (pI) or molecular mass.

+ _

_ _ _ _ + + + +

+ _

314

Strecker Synthesis: recall reductive amination

C CO2HR

O NH3

C CO2HR

NH2

H

NaB(CN)H3

R C CO2H

NH2

H

C HR

O NH3

C HR

NH2NaC!N

N!C:

R C C!N

NH2

H

H3O+

-or-NaOH, H2O

R C CO2H

NH2

H

Amidomalonate Synthesis: recall the malonic acid synthesis

H CO2EtC

CO2EtHN EtO Na

RCH2X

H3O

- CO2 RCH2 CO2HC

HH2N

O

RCH2 CO2EtC

CO2EtHN

O

R-CH2-CO2H R C CO2H

Br

R C CO2H

NH2Br2, PBr3 NH3

H H

27.5: Synthesis of Amino Acids:

Ch. 19.16

161

315

R CO2

H3N H H3C O CH3

O O

base R CO2H

HN H

O

H3C

HOCH2CH3

H+R CO2CH2CH3

H2N H

27.6: Some Biochemical Reactions of Amino Acids. Manyenzymes involved in amino acid biosynthesis, metabolism and catabolism are pyridoxal phosphate (vitamin B6) dependent (please read)

NH3

R CO2-

H

L-amino acid

NH3

R HCO2

-

D-amino acid

racemase,epimerase

H3N

RH

H

decarboxylase

O

R CO2-

transaminase

N

2-O3POOH

O H

pyridoxalphosphate (PLP)

+

NH

PO

N

OH

CO2-R

27.5: Reactions of Amino Acids. Amino acids will undergoreactions characteristic of the amino (amide formation) and carboxylic acid (ester formation) groups.

316

27.7: Peptides. Proteins and peptides are polymers made up of amino acid units (residues) that are linked together through theformation of amide bonds (peptide bonds) from the aminogroup of one residue and the carboxylate of a second residue

By convention, peptide sequences are written left to right from the N-terminus to the C-terminus

H2N CO2H

Alanine

H2N CO2H

HO

Serine

+ - H2O

H2N

HN

O

CO2H

OH

Ala - Ser(A - S)- H2O

H2N

HN

O

CO2H

HO

Ser - Ala(S - A)

C-terminus

N-terminus

C-terminus

N-terminus

NH

O

R3

HN

O

R2

NH

O

R1

HN

NH

O

R7

HN

O

R6

NH

O

R5

R4 HN

O

backbone

162

317

The amide (peptide) bond has C=N double bond character dueto resonance resulting in a planar geometry

restricts rotationsresistant to hydrolysis

R1

HN

N

O R2 HN

O

amide bond

R1

HN

N

O R2 HN

O

_

+

H H

The N-H bond of one amide linkage can form a hydrogen bondwith the C=O of another.

N

N

O

H

R

O

H

N

N

O

H

R

O

H

N

N

O

H

R

O

H

N-O distance 2.85 - 3.20 Å optimal N-H-O angle is 180 °

Disulfide bonds: the thiol groups of cysteine can be oxidized to form disulfides (Cys-S-S-Cys)

SHHO2C

NH22

1/2 O2H2O

SHO2C

NH2

SCO2H

NH2H2

318

NH

O

R2

HN

O

R1

NH

HN

O

R5

NH

O

R4

HN

O

NH

OHN

O

R6

NH

HN

O

R10

NH

O

R9

R8 HN

O

SH

NH

O

R2

HN

O

R1

NH

HN

O

R5

NH

O

R4

HN

O

NH

OHN

O

R9

NH

HN

O

R13

NH

O

R12

R11 HN

O

S

SHS 1/2 O2

H2

1986 Nobel Prize in Medicine:Stanley Cohen Rita Levi-Montalcini

Epidermal Growth Factor (EGF): the miracle of mother’s spit 53 amino acid, 3 disulfide linkages

163

319

27.8: Introduction to Peptide Structure Determination.Protein Structure:primary (1°) structure: the amino acid sequencesecondary (2°): frequently occurring substructures or foldstertiary (3°): three-dimensional arrangement of all atoms in a

single polypeptide chainquaternary (4°): overall organization of non-covalently linked

subunits of a functional protein.

1. Determine the amino acids present and their relative ratios2. Cleave the peptide or protein into smaller peptide fragments

and determine their sequences3. Cleave the peptide or protein by another method and

determine their sequences. Align the sequences of thepeptide fragments from the two methods

320

E-A-Y-L-V-C-G-E-RF-V-N-Q-H-L-F-S-H-L-KG-C-F-L-P-KL-G-A

F-V-N-Q-H-L-FS-H-L-K-E-A-YL-V-C-G-E-R-G-C-FL-P-K-L-G-A

F-V-N-Q-H-L-F F-V-N-Q-H-L-F-S-H-L-K S-H-L-K-E-A-Y E-A-Y-L-V-C-G-E-R L-V-C-G-E-R-G-C-F G-C-F-L-P-K L-P-K-L-G-A

L-G-A

F-V-N-Q-H-L-F-S-H-L-K-E-A-Y-L-V-C-G-E-R-G-C-F-L-P-K-L-G-A

164

321

27.9: Amino Acid Analysis. automated method to determine the amino acid content of a peptide or protein

Reaction of primary amines with ninhydrin

peptide-or-

protein

[H] reduce anydisulfidebonds

Enzymaticdigestion

R CO2

NH3 individualamino acids-or-

H3O+, Δ

1972 Nobel Prize in ChemistryWilliam Stein Stanford Moore

liquidchromatography

derivatize w/ninhydrin

Detected w/UV-vis

Different amino acids have different chromatographicmobilities (retention times)

R CO2

NH3

O

O

O+

Ninhydrin

O

O

N

O

O

+ RCHO + CO2

322

27.10: Partial Hydrolysis of Peptides. Acidic hydrolysis ofpeptides cleave the amide bonds indiscriminately.

Proteases (peptidases): Enzymes that catalyzed the hydrolysisof the amide bonds of peptides and proteins.

Enzymatic cleavage of peptides and proteins at defined sites:• trypsin: cleaves at the C-terminal side of basic residues,

Arg, Lys but not His

• chymotrypsin: cleaves at the C-terminal side of aromatic residuesPhe, Tyr, Trp

NH

O

HN

O

NH

R3

O

HN

R1O

H3NCO2

NH3

NH

O

HN

O

H3N

R3

O

HN

R1O

H3NCO2

NH3

O +trypsin

H2O

NH

O

HN

O

NH

R3

O

HN

R1O

H3NCO2 N

HO

HN

O

H3N

R3

O

HN

R1O

H3NCO2

O +chymotrypsin

H2O

165

323

Trypsin and chymotrypsin are endopeptidases

Carboxypeptidase: Cleaves the amide bond of the C-terminal amino acid (exopeptidase)

27.11: End Group Analysis. The C-terminal AA is identified by treating with peptide with carboxypeptidase, then analyzingby liquid chormatography (AA Analysis).

N-labeling: The peptide is first treated with 1-fluoro-2,4-dinitrobenzene (Sanger’s reagent), which selectively reacts with the N-terminal amino group. The peptide is then hydrolyzed to theiramino acids and the N-terminal amino acid identified as itsN-(2,4-dinitrophenyl) derivative (DNP).

NO2

O2N

F

H3N

O

R1 HN CO2+

!

NH

O

R1 HN CO2nucleophilic

aromaticsubstitution

NO2

O2N

NH

O

R1

NH2

NO2

O2N

+plus other unlabeled

amino acids

enzymatic

digestion

-or-

H3O+, !

324

27.12: Insulin. (please read) Insulin has two peptide chainsInsulin has two peptide chains (the A chain(the A chain has 21 amino acids and the B chain has 30 aminohas 21 amino acids and the B chain has 30 amino acids) held together by two disulfide linkagesacids) held together by two disulfide linkages

Pepsin: cleaves at the C-terminal side of Phe, Tyr, Leu; butnot at Val or Ala

Pepsin cleavageTrypsin cleavageH3O+ cleavage

166

325

27.13: The Edman Degradation and Automated PeptideSequencing. Chemical method for the sequential cleavage andidentification of the amino acids of a peptide, one at a time starting from the N-terminus. Reagent: Ph-N=C=S, phenylisothiocyanate (PITC)

S

C

NPh

+H2N

O

R1 HN CO2

pH 9.0

NH

O

R1 HN CO2

NH

S

Ph

H+

H+

HN

S

OH

HN CO2

N

R1

PhH+

HN

S OH2N CO2

N

R1

Ph

H+

HN

N OS

R1

Ph

+

N-phenylthiohydantoin:separated by liquid chromatography (based of the R group) and detected by UV-vis

-1 peptide with a new N-terminal amino acid (repeat degradation cycle)

326

Peptide sequencing by Edman degradation: • Cycle the pH to control the cleavage of the N-terminal amino acid by PITC. • Monitor the appearance of the new N-phenylthiohydantoin for each cycle. • Good for peptides up to ~ 25 amino acids long. • Longer peptides and proteins must be cut into smaller fragments before Edman sequencing.

Tandem mass spectrometry has largely replaced Edman degradation for peptide sequencing

27.14: The Strategy for Peptide Synthesis:Chemical synthesis of peptide:

1. Solution phase synthesis2. Solid-phase synthesis

167

327

H2N CO2H

Ala

H2N CO2H

Val

+- H2O

H2N

HN

O

CO2H

Ala - Val(A - V)

H2N

HN

O

CO2H

Val - Ala(V - A)

- H2O

The need for protecting groups

Orthogonal protecting group strategy: the carboxylate protecting group must be stable to the reaction conditions for the removal of the α-amino protecting group and ( vice versa)

NH

Ala

H2N

Val

+

- H2O

NH

HN

O

Ala - Val(A - V)

Pn OH

O

OPc

O

PnOPc

Oselectivelyremove Pn

H2N

HN

O

Ala - Val(A - V)

OPc

O

peptidecoupling

peptidecoupling(-H2O)

NH

Pn OH

O

Ph

Phe (F)

NH

HN

O

OPc

OOHN

Ph

Pn

Phe - Ala - Val(F - A - V)

Repeat peptide synthesis

328

27.15: Amino Group Protection. The α-amino group is protected as a carbamate.

27.16: Carboxyl Group Protection. Protected as a benzylester; removed by hydrogenolysis

RO Cl

O+ Base

NH

O

O

O

tert-butoxycarbonyl(t-BOC)

removed withmild acid

R

NH

O

O

O

C6H5

benzyloxycarbonyl(cBz)

removed with mild acid or by hydrogenolysis

R

NH

O

O

O

fluorenylmethylcarbonyl(FMOC)

removed with mild base(piperidine)

NH3

O

O NH

O

RO

O

OH

NH

H2N+- H2O

OH

O

O

O

peptidecoupling

O

OC6H5

C6H5NH

HN

O

O

O C6H5

O

OC6H5

mild acid

H2N

HN

O

O

O C6H5

OH

O

Ph

NH

O

OC6H5

- H2Opeptidecoupling

NH

HN

O

OOHN

Ph

O C6H5

O

OC6H5

H2, Pd/C

NH

HN

O

O

OO

H3N

Ph

168

329

27.17: Peptide Bond Formation. Amide formation from the reaction of an amine with a carboxylic acid is slow. Amide bondformation (peptide coupling) can be accelerated if the carboxylicacid is activated. Reagent: dicyclohexylcarbodiimide (DCC)

C6H11 N C N C6H11

R O

O

C6H11 N C NH

C6H11

R O

O

+

R O

O

C

N

C6H11

C6H11

NH

R'-NH2

••

R O

O

C

HNC6H11

C6H11

NH

NHR' +

R NH

O

R'NH

NH

O

C6H11 C6H11+

(DCC)

Amide DCU

"activated acid"

R O

O

C

N

C6H11

C6H11

NH

NR'

++H

H

H

NH

Ala

H2N

Val

+NH

HN

OcBz OH

O

OBn

O

cBzOBn

O

selectivelyremove N-protecting

group

H2N

HN

O

OBn

O

NH

cBz OH

O

Ph

Phe (F)

NH

HN

O

OBn

OOHN

Ph

cBz

DCC

peptidecoupling

CF3CO2H

DCCH2, Pd/C

NH

HN

O

OH

OO

H2N

Ph

Phe - Ala - Val(F - A - V)

330

• In order to practically synthesize peptides and proteins, time consuming purifications steps must be avoided until the very end of the synthesis.• Large excesses of reagents are used to drive reactions forward and accelerate the rate of reactions.• How are the excess reagents and by-products from the reaction, which will interfere with subsequent coupling steps, removed without a purification step?

27.18: Solid-Phase Peptide Synthesis: The Merrifield Method.Peptides and proteins up to ~ 100 residues long are synthesized on a solid, insoluble, polymer support. Purification is convenientlyaccomplished after each step by a simple wash and filtration.

169

331

The solid support (Merrifield resin): polystyrene polymer

H2N

Val

O

O

peptidecoupling

NH

FMOC OH

ODCC

O

O

HN

FMOC NH

Opurify:

wash & filter

remove N-protecting

group

O

O

H2NNH

O

NH

FMOC OH

O

Ph

Phe (F)

DCC

O

O

NH

O

purify:wash & filter

NH

FMOCHN

O

Ph purify:wash & filter HF

remove N-protecting

group and cleavefrom solid-support

OH

O

NH

O

H2N

HN

O

Phpurify by liquidchromatograrphy

or electrophoresis

NH

remove N-protecting

group

NH

Solid-phase peptide synthesis

O

R

OHN_

O

O

R

NH

CF3CO2H

O

O

R

NH2

BOC

BOC

+

polymerization

styrene

divinylbenzene(crosslinker, ~1 %)

initiator

Ph

Ph Ph Ph Ph

PhPhPh Ph

Ph

Ph

Ph

Ph

Ph

H3COCH2ClZnCl2

CH2Cl

332

LYS GLU THR ALA ALA ALA LYS PHE GLU ARG GLN HIS MET ASP SER SER THR SER ALA ALA SER SER SER ASN TYR CYS ASN GLN MET MET LYS SER ARG ASN LEU THR LYS ASP ARG CYS LYS PRO VAL ASN THR PHE VAL HIS GLU SER LEU ALA ASP VAL GLN ALA VAL CYS SER GLN LYS ASN VAL ALA CYS LYS ASN GLY GLN THR ASN CYS TYR GLN SER TYR SER THR MET SER ILE THR ASP CYS ARG GLU THR GLY SER SER LYS TYR PRO ASN CYS ALA TYR LYS THR THR GLN ALA ASN LYS HIS ILE ILE VAL ALA CYS GLU GLY ASN PRO TYR VAL PRO VAL HIS PHE ASP ALA SER VAL

His-12 AHis-119 A

His-12 BHis-119 B

pdb code: 1AFL

Ribonuclease A- 124 amino acids, catalyzes the hydrolysis of RNASolid-phase synthesis of RNase A:

Synthetic RNase A: 78 % activity 0.4 mg was synthesized 2.9 % overall yield average yield ~ 97% per coupling step

R. Bruce Merrifield, Rockefeller University, 1984 Nobel Prize in Chemistry: “for his development of methodology for chemical synthesis on a solid matrix.”

170

333

27.19: Secondary Structures of Peptides and Proteins.β-sheet: Two or more extended peptide chain, in which the amidebackbones are associated by hydrogen bonded

parallel

crossover

N

O

R

N

O

R

N

N

O

R

HN

O

R

N

O

R

O

R

N

R

O

O

H

H

H

H

H

H

N

O

R

N

O

R

N

N

O

R

HN

O

R

N

O

R

O

R

N

R

O

O

H

H

H

H

H

H

N → C

N → C

N → C

C ← N

anti-parallel loopor

turnN

O

R

N

O

R

N

N

O

R

N

O

R

N

O

R

O

R

N

R

O

O

H

H

H

H

H

H

N

O

N

O

R

N

N

O

R

N

O

R

N

O

R

O

R

N

R

O

O

H

H

H

H

H

H H

HN → C

N → C

C ← N

N → C

334

α-helix: 3.6 amino acids per coil, 5.4 Å

3.6 AA5.4 Å

C

N

171

335

myoglobin pdb code: 1WLA

Anti-parallel β-sheets of lectin

pdb code: 2LAL

Bacteriorhodopsinpdb code: 1AP9

Parallel β-sheets carbonic anhydrase

pdb code: 1QRM

336

27.20: Tertiary Structure of polypeptides and Proteins.Fibrous. Polypeptides strands that “bundle” to form elongated fibrous assemblies; insoluble;

Globular. Proteins that fold into a “spherical” conformation .

Hydrophobic effect. Proteins will fold so that hydrophobic amino acids are on the inside (shielded from water) and hydrophilic amino acids are on the outside (exposed to water).

Pro • Ile • Lys • Tyr • Leu • Glu • Phe • Ile • Ser • Asp • Ala • Ile • Ile • His •Val • His • Ser • Lys

172

337

Enzymes: proteins that catalyze biochemical reactions.• by bringing the reactive atoms together in the optimal geometry for the reaction.• lowering the activation energy (ΔG‡) by stabilizing the transition state and/or high energy intermediate. • many enzymes use the functional groups of the amino acid sidechain to carry out the reactions

Proteases (peptidases): catalyzes the hydrolysis of peptide bonds

NH

HN

NH

O

RO

HN

N

O R

RO

R

H

O protease

H2OH3N

CO2 NH

HN

H3N

O

RO

HN

N

O R

RO

R

H

O

H3NCO2O +

Four classes of proteases:Serine (trypsin): aspartate-histidine-serine Aspartyl (HIV protease, renin): two aspartatesCysteine (papain, caspase): histidine-cysteineMetallo (Zn2+) (carboxypeptidase, ACE): glutamate

338

Mechanism of carboxylpeptidase, metalloprotease (p. 1151)Mechanism of a serine protease (trypsin, chymotrypsin):

OSer195

H

N

NHis57

R1 NH

O

R2

NH HN

oxy-anionhole

H

Asp102 CO2-

R1 NH

O

R2

NH HN

OSer195

N

NHis57

H

Asp102 CO2-

H- R2-NH2

Ser192O R1

O

NH HN

acyl-enzymeintermediate

OH

H

N

NHis57

H

Asp102 CO2-

Ser192O R1

O

NH HN

OH

N

NHis57

H

Asp102 CO2-

H

OSer195

H

N

NHis57

H

Asp102 CO2-

RCO2H+

173

339

27.21: Coenzymes. Some reactions require additional organic molecules or metal ions. These are referred to as cofactors orcoenzymes.

N N

SOP

HO O

O POH

OH

O

NH2

N

+

Thiamin Diphosphate(vitamin B1)

OP

OHO

HO

N

O

OH

H

Pyridoxal Phophates(vitamin B6)

N

NN

N

O

HOO

OH

Fe

Heme

S

NHHN

O

CO2H

Biotin(vitamin B7)

NN

N N

H2N

O NH2

O

NH2

NH2O

O

O

Co

O

H

H2N

O

NH

O

C

PO

O

-OO

N

N

HO H

H

NH2

H

H

OH

N

Vitamin B12

(cyanocobalamin)

HN

N

N

N

HN

H2N

O

HN

O CO2-

CO2-

Folic Acid(vitamin B9)

N

N

NH

N O

O

O

OH

HO

OH

O-

P

O

P O

O

O-

O

HO

N

OH

O

N

N

N

NH2

Flavin Adenine Diphosphate (FAD)(Vitamin B2)

27.22: Protein Quaternary Structure. (please read)